Vaccination is intended to increase an individual's subsequent responsiveness to pathogens or tumor antigens. Increased humoral responsiveness is dependent on both increased TH function and the generation of a population of memory B cells whose sIg receptors are selected for high affinity for the immunizing antigen. The goal of this project is to de fine the parameters of the generation and propagation of memory B cells in order to provide basic information essential to future efforts to maximize this process. For this purpose, we will capitalize on: 1) the isolation of a B cell subset (HSA1o) from naive mice that is enriched for memory progenitors, and 2) our ability to generate memory B cells in vitro. Because in vitro (fragment culture) technology facilitates t he manipulation of the responding cells and stimulatory environment, as well as enabling the assessment of: clonal expansion, the acquisition of phenotypic markers prototypical of memory B cells, the accumulation of somatic mutations, affinity maturation and the responsiveness of newly generated memory B cells, we believe the proposed experiments will provide an unique opportunity to address numerous outstanding questions concerning memory B cell generation. These include; a) How are somatic hypermutation, affinity maturation and clonal expansion regulated and are these linked or independent processes? b) Do precursors of naive B cell subsets other than HSA 1o cells have the capacity to generate memory B cells? c) How is B cell memory propagated and are there cells within the memory B cell population that can further somatically mutate and give rise to subsequent generations of memory cells? d) What is the capacity of cells of various T cells subsets to participate in memory B cell generation? e) Is there a need for the persistence of antigen and/or TH during he course of memory B cell clonal progression? f) What is the role of antibody and antigen-antibody complexes in memory cell generation and affinity maturation? and g) What is the requirement for various cytokines and co-receptor (CD40-CD40 ligand) interactions in memory B cell clonal development and isotype commitment: We anticipate that the proposed in vitro analyses of memory B cell generation in concert with studies of memory T cell generation in Projects 1 and 2, and the proposed collaborative studies wherein interactions of T and B cells specific for cyt c and influenza virus are analyzed, will provide information that will further our basic understanding of the generation and regulation of memory responses and thus facilitate the future enhancement of vaccination protocols.